The wild-type p53-induced phosphatase Wip1 (PPM1D) is a member of the serine/threonine protein phosphatase 2C (PP2C) family. Although Wip1 is expressed at low levels in most normal cells, its transcription is induced by p53 after exposure of cells to DNA damage-inducing agents, such as ionizing radiation (IR) or ultraviolet (UV) light. The Wip1 protein is frequently overexpressed or the PPM1D gene is frequently amplified in several human cancers, and this increased expression is associated with worse outcomes. Studies of human cells have shown that overexpression of Wip1 compromises tumor suppressor functions, and studies of mice that lack Wip1 show that they are resistant to tumorigenesis. The current research on Wip1 is focused on understanding its regulation and functions, identifying its functional targets and performing high-throughput screens (HTS) to identify specific inhibitors/activators of Wip1 phosphatase activity. Recently, we characterized the effects of the binding of the labile metal ion and the phospho-peptide substrate on the conformation of human PPM1A, a family member of Wip1, by both hydrogen/deuterium exchange mass spectrometry and x-ray crystallography. Together, these structural studies have allowed us to better understand substrate binding in this family of phosphatases and to characterize the labile third metal ion that is essential for catalytic activity, both critical aspects that could be abrogated by the binding of a specific inhibitor. A high-resolution structure of the Wip1 catalytic domain that includes the conformation of the B-loop would greatly aid further development of specific inhibitors of Wip1 phosphatase activity. Additionally, high-resolution structural information for the Wip1 catalytic site would be useful for further optimization of known inhibitors and to guide structure-activity investigations of inhibitors or activators identified in HTS studies. We have expressed a SUMO-rWip1 (1-403) fusion protein that has improved solubility in E. coli while maintaining wild-type catalytic activity. The SUMO tag can be removed to restore the native N-terminal sequence. We have prepared milligram amounts of highly purified protein to initiate crystallization experiments. In collaboration with Drs. Fred Dyda and Dalibor Kosek, Laboratory of Molecular Biology, NIDDK, we have made several attempts to obtain crystals using rWip11-403 alone, in complex with GSK2830371, an allosteric Wip1 inhibitor, or in complex with our optimized cyclic peptide inhibitor. Very recently, we obtained crystals and are proceeding to solve the structure. In collaboration with Dr. Barbara Stecca (ISPRO, Florence, Italy) we found that CDK1 kinase decreases the transcriptional activity of the Hedgehog pathway and decreases GLI1 protein levels, in opposition to Wip1, which enhances GLI1 activity by dephosphorylating GLI Thr1045, demonstrating that inhibition of Wip1 may be an effective therapeutic approach for human cancers that overexpress Wip1 and have activated Hedgehog signaling. In collaboration with Dr. Oleg Demidov (University of Burgundy, Dijon, France), we are using syngeneic tumor models to investigate the effects on tumor progression of ablating Wip1 in the immune system. In syngeneic mouse models, myeloid-specific deletion of Wip1 delayed the growth of both B10 melanoma tumors and LLC1 lung cancer tumors, confirming an important role of Wip1-deficient innate immune cells in anti-tumor immunity and suggesting that Wip1 is a promising target for increasing the efficiency of anti-cancer immunotherapy. Wip1 is amplified or overexpressed in numerous human cancers including breast cancer, ovarian clear cell carcinoma, gastric cancer, pancreatic adenocarcinoma, medulloblastoma, and neuroblastoma. Thus, developing inhibitors of Wip1 activity may be beneficial in the treatment of several human cancers. We have developed and validated two orthogonal plate-based Wip1 activity assays for HTS. The two assays have high sensitivity and broad dynamic range enabled by fluorescence detection or mass spectrometry and are suitable for screening compound libraries for modulators of Wip1 activity. We propose to screen a diverse set of compound libraries at the National Center for Advancing Translational Sciences (NCATS). Together, these libraries provide an appealing combination of diverse chemical scaffolds and well-characterized compounds possessing excellent properties for further therapeutic development.